We seek to characterize the cis-regulatory elements controlling Th1 versus Th17 differentiation. Specific Aims: 1) Identify cis-regulatory elements contributing to differential expression of Th1 and Th17 lineage genes. 2) Identify alterations in chromatin looping in the Il17a/f, H12rb2/ll23r, and N22/lfng gene loci associated with the Th1 or Th17 fate. 3) Determine the importance of Th1 and Th17 lineage-specific TFs in chromatin modifications, looping and gene expression. Hypothesis: 1) In Th17 but not in Th1 cells, shared cis-regulatory elements will be brought in close contact with the promoters of Il17a and Il17f through chromatin looping, and this looping and coordinate expression of these genes will be dependent on binding of Th17 lineage-specifying (TFs) to these shared elements. 2) Alternative rather than coordinate expression of N12rb2 vs. H23r and Ifng vs. II22 by Th1 and Th17 cells will reflect differential utilization of distal regulatory elements and chromatin looping in these two loci, and be attributable to Th1 and Th17 lineage-specific TFs binding to alternative cis-regulatory elements or competing for binding to shared elements. Approach: We will use the complementary approaches of genome-wide DNase I hypersensitivity assay and chromatin immunoprecipitation coupled to microarray (ChlP-chip) on naive murine CD4+ T lymphocytes and in vitro polarized Th1 and Th17 cells to identify regulatory elements involved in the regulation of the IL17a/f, IL12rb2/N23r and N22/lfng gene loci. Chromatin looping will be investigated in parallel by chromatin conformation capture (3C) assay. The influence of lineage-specific TFs on chromatin looping, histone modifications and gene expression will be assessed by 3C, ChIP, and qPCR in lymphocytes from mice with genetic deletions of TFs critical to Th1 or Th17 fate. Relevance: The Th17 cytokines IL-17 and IL-22 have emerged as important mediators of chronic inflammatory bowel diseases and evidence from the literature suggests that they may also mediate the increased potential for oncogenic transformation seen in Crohn's disease and ulcerative colitis. Given the importance for Th1 cells and their effector cytokine Ifng in tumor surveillance and clearance, and the potential for Th17 cells to foster tumor development, elucidation of those regulatory elements that drive the Th17 and Th1 differentiation is an important goal. Understanding the mechanisms promoting commitment to the Th17 versus Th1 lineages at the epigenetic level may allow us to subvert lineage decisions to alleviate chronic inflammation and reduce the associated cancer risk.